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Cellular Id1 inhibits hepatitis B virus transcription by interacting with the novel covalently closed circular

Jie Wei^1,2, Yueyuan Shi^3,4, Chunhong Zou³

¹Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China.

International Journal of Biological Sciences

|January 3, 2022

Abstract:

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), which required developing novel therapies targeting the inhibition of HBV transcription and replication due to current limited treatment options. We explored novel target for the development of novel therapies targeting the inhibition of HBV replication and transcription. The expression of Id1 and E2F4 in HCC cells and tissues was detected by qRT-PCR and western blot. We investigated the Id1 and E2F4-mediated transcription of HBV infection by using HepG2.2.15, HepAD38, HepG2-NTCP cell lines and AAV/HBV-infected mice. Interactions between the two host proteins and viral covalently closed circular DNA (cccDNA) were assessed using subcellular localization, protein-protein interaction, chromatin immunoprecipitation, and luciferase assays. Ectopic Id1 significantly reduced HBV transcription and replication in both HBV-expressing cells and AAV/HBV-infected mice. Id1 and E2F4 could form a heterodimer to prevent E2F4 from promoting HBV transcription and replication. E2F4 could directly bind to cccDNA and activate the HBV core promoter in cell lines. Furthermore, in vitro binding experiments confirmed that the sequence 1758'-TTAAAGGTC-1766', which is highly conserved among HBV genotypes, is the target site of the E2F4 homodimer. The findings suggest that E2F4 function as novel cccDNA-binding protein to directly activate HBV transcription by binding to Cp promoter region. Our results highlight the ability that E2F4 represent a pan-potential therapeutic target against HBV transcription and provide more clues to better understand the life cycle of HBV.

Insights

Researchers identified E2F4 as a key protein that activates Hepatitis B virus (HBV) transcription by binding to its cccDNA. This finding offers a potential new therapeutic target for treating HBV infection.

Area of Science:

Hepatology
Virology
Molecular Biology

Background:

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC).
Current treatments for HBV are limited, necessitating novel therapeutic strategies targeting viral transcription and replication.
Host factors influencing HBV replication are crucial for understanding viral pathogenesis and developing new therapies.

Purpose of the Study:

To identify and characterize novel host factors involved in regulating Hepatitis B virus (HBV) transcription and replication.
To investigate the role of Id1 and E2F4 in HBV infection and their potential as therapeutic targets.
To elucidate the mechanism by which E2F4 interacts with HBV cccDNA to modulate viral gene expression.

Main Methods:

Quantitative real-time PCR (qRT-PCR) and Western blot to assess Id1 and E2F4 expression.
Utilized HepG2.2.15, HepAD38, and HepG2-NTCP cell lines, along with AAV/HBV-infected mice for in vivo studies.
Employed subcellular localization, protein-protein interaction, chromatin immunoprecipitation, and luciferase assays to study protein-cccDNA interactions and transcriptional activity.

Main Results:

Ectopic expression of Id1 significantly inhibited HBV transcription and replication in vitro and in vivo.
Id1 and E2F4 form a heterodimer, with Id1 impeding E2F4's role in promoting HBV transcription.
E2F4 directly binds to a conserved sequence on HBV cccDNA, activating the viral core promoter and thus viral transcription.

Conclusions:

E2F4 acts as a novel cccDNA-binding protein that directly activates HBV transcription via the Cp promoter region.
E2F4 represents a potential pan-therapeutic target for inhibiting HBV transcription across various genotypes.
Understanding E2F4's role provides new insights into the HBV life cycle and potential avenues for antiviral therapy.

Read full analysis

Keywords:

E2F4 HBV Id1 cccDNA promoter.

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